Neuroinflammation occurs in both acute and chronic neurodegenerative disorders and contributes to neural injury. Microglial cells are activated in the inflammatory response, releasing cytokines, nitric oxide (NO), and superoxide which target neighboring neurons. The overwhelming majority of studies investigating neurodegenerative disease mechanisms in vitro are conducted at atmospheric O2 despite the fact that this pO2 (21%, 160 mm Hg) far exceeds physiological brain pO2 (~3%, 23 mm Hg). We propose that pO2 fundamentally influences the mechanisms of neuronal injury and impacts the success of therapeutic strategies. We found that at 3% O2, NO-mediated inhibition of cortical neuron respiration is over 10-fold more potent than at atmospheric O2, and, in contrast to current dogma, occurs without inhibition of complex IV but with inhibition of complex II. This study will test the central hypothesis that at brain physiological O2, NO derived from microglial inducible nitric oxide synthase (iNOS) causes cortical neuron injury by S-nitrosylation-mediated inactivation of succinate dehydrogenase upstream of complex III rather than by NO or peroxynitrite-mediated inhibition of complex IV. Clinically safe idebenone, a short chain Coenzyme Q analogue which upon reduction by NQO1 enzyme can shuttle electrons from cytoplasmic NAD(P)H directly to complex III, will be tested for the ability to bypass an upstream respiratory impairment, rescue ATP levels, and improve neuronal survival at either 3% or 21% O2. We predict that drugs that induce the Nrf2/antioxidant response element pathway will potently synergize with idebenone by 1) upregulating NQO1 and by 2) increasing the pool of the endogenous antioxidant glutathione which can prevent cysteine S-nitrosothiol modifications. In aim 1 we will determine how pO2 influences the mechanism of neurotoxicity exerted by activated microglia. In aim 2 we will determine whether exogenous NO impairs mitochondrial function in cortical neurons at physiological (3%) O2 by reversible S-nitrosylation-mediated inhibition of succinate dehydrogenase (complex II). In aim 3 we will determine whether idebenone, when combined with an inducer of the Nrf2/antioxidant response element pathway, sulforaphane, can bypass NO-mediated respiratory inhibition and prevent microglial toxicity. We will for the first time perform neuronal respiration measurements at physiological 3% O2 using the Seahorse XF24 and also adapt this technology for examining neuronal respiratory impairments caused by co-cultured, activated microglia. Our long-term goals are to elucidate mitochondrial mechanisms of neural injury and identify new avenues for therapeutic intervention. Here, we will take important steps toward these goals by 1) determining how pO2 influences neuronal injury, with the potential for a paradigm-shifting change in the way in vitro neurodegenerative research is conducted, and by 2) providing proof-of-principle on whether bypass of respiratory inhibition is a viable strategy to reduce microglial neurotoxicity.